Mucolipin-3 (MCOLN3) is a pH-regulated calcium channel that localizes to the endosomal pathway. Gain-of-function mutation in MCOLN3 causes the varitint-waddler (Va) phenotype in mice, which is characterized by hearing loss, vestibular dysfunction, and coat color dilution. The Va phenotype results from a punctual mutation (A419P) in the pore region of MCOLN3 that locks the channel in an open conformation causing massive entry of calcium inside cells and inducing cell death by apoptosis. Overexpression of wild-type MCOLN3 produces severe alterations of the endosomal pathway, including enlargement and clustering of endosomes, delayed EGF receptor degradation, and impaired autophagosome maturation, thus suggesting that MCOLN3 plays an important role in the regulation of endosomal function. To understand better the physiological role of MCOLN3, we inhibited MCOLN3 function by expression of a channel-dead dominant negative mutant (458DD/KK) or by knockdown of endogenous MCOLN3 and measure several endosomal parameters including luminal calcium, pH, and endosomal fusion. Luminal calcium concentration and pH were measured by fluorescence ratio imaging. To monitor calcium, cells were simultaneously loaded cell-impermeant Oregon Green 488 BAPTA-5N (calcium indicator) and Alexa Fluor 555-conjugated dextran (nonsensitive to calcium), while measurement of endosomal pH was performed by simultaneous loading of cells with FITC-conjugated dextran (sensitive to pH) and Alexa Fluor 555-conjugated dextran (not sensitive to pH). The ratio of green to red fluorescence is indicative of the luminal calcium concentration and pH in endocytic vesicles. Finally, we developed an in vitro assay to measure homotypic fusion of endosomes. Briefly, one population of cells was incubated with Alexa-Fluor-488 EGF, and the other cells were incubated with Alexa-Fluor-555 EGF for 15 min to label the early endosomal compartment. Endosomes were then purified and subjected to an in vitro fusion assay. At the end of the incubation, endosomes were fixed on glass coverslips and analyzed by confocal microscopy. Endosomal fusion was quantified by measuring the proportion of yellow endosomes. We found impairment of MCOLN3 activity caused a significant accumulation of luminal calcium at endosomes. This accumulation led to severe defects in endosomal acidification as well as to increased endosomal fusion. Our findings reveal a prominent role for MCOLN3 in regulating calcium homeostasis at the endosomal pathway and confirm the importance of luminal calcium for proper acidification and membrane trafficking. Future experiments will address the role of MCOLN3 in animal models.